(pt 2) exam #1 - intro to molecular diagnostics (cls 605)

PCR + qPCR

polymerase chain reaction (PCR)

in vitro DNA replication

• A way of selectively replicating a particular segment of DNA in a complex mixture of DNA

• The segment to be copied is identified for the DNA polymerase by primers

• useful in research, making specific proteins, infectious disease, genetic diseases, forensics

basic ingredients for PCR

• Heat stable DNA polymerase (Taq polymerase)

• DNA nucleotides: DNA building blocks

• Primers: short ssDNA

• Template DNA: DNA to be copied

• Reaction buffer--contains magnesium ions (cofactor for enzymes)

components of standard PCR reaction mix

• 0.25 mM each primer

• 25 mM each dATP, dCTP, dGTP, dTTP

• 50 mM KCl

• 10 mM Tris, pH 8.4 (buffer)

• 1.5 mM MgCl2

• 2.5 units polymerase

• 102-105 copies of template

• 50 uL reaction vol total

PCR master mix

• mixture of all the ingredients of PCR minus the template

  • Benefits: more efficient, less room for error

• Multiply all the amounts of each ingredient by the number of reactions you are going to run (plus extra)

• Distribute to each individual reaction tube before adding template

(PCR ingredients) Taq polymerase

isolated from hot water bacterium Thermus aquaticus

(PCR ingredients) deoxynucleotides

• dATP

• dTTP

• dGTP

• dCTP

(PCR ingredients) primers

• Synthetic single stranded DNA between 18 and 50 nucleotides long (oligonucleotides)

• Necessary because:

  • Need to define DNA sequence to be copied

  • DNA pol needs 3' OH to add nucleotide to

types of primers for PCR

• forward and reverse

  • Forward primer (5’→3’) = binds to antisense strand (3’→5’)

  • Reverse primer (3→5’) = binds to the sense/coding strand (5’→3’)

• Flank sequence of interest

• Each primer is complementary to a different strand

(PCR ingredients) buffer

• Tris-HCl: maintains appropriate pH

• Salts:

  • KCl--stabilize DNA, promote primer annealing

  • MgCl2--Mg is a cofactor for Taq pol

• Other stabilizers and enhancers: DMSO, BSA

PCR cycle/steps (general)

• Step 1: denaturation (~94 deg C)

  • dsDNA becomes single stranded

• Step 2: annealing (depends on primers, 54-60 C)

  • Primers bind to template

• Step 3: extension (depends on pol, 72 C)

  • Polymerase synthesizes complementary strand

• Repeated for 30-40 cycles

  • exponential amplification

qualitative vs quantitative PCR

• Qualitative: electrophoresis and staining

  • answers if there is DNA or not

• Quantitative: qPCR (aka real-time PCR)

  • Can determine viral load

interpreting PCR results

• The PCR product should be of the expected size (or melting point)

• No product should be present in the reagent blank

• Misprimes may be seen due to non-specific annealing of primers

  • Primers stick to some sequence nonspecifically and start making a product

• Primer dimers may be seen due to annealing of primers to each other

optimizing PCR (general)

• Critical PCR parameters

  • Primer design

  • Annealing temperature

  • Concentration of DNA template, nucleotide, divalent cations (Mg2+)

  • Type of polymerase (hot start enzymes require initial heat activation)

(optimizing PCR) primer design

• CRITICAL for successful PCR

  • Make sure that your primers are at the correct location to amplify sequence of interest

  • Primers should be specific (i.e. don't want primers that target things that are repetitive)

  • Should avoid primers that anneal to each other

  • Primer melting temperature

(primer design) specificity/length of primer

• Probability that any particular oligo sequence will occur randomly in a template decrease with length

• If primers are too long:

  • Poor annealing

  • More likely to bind to each other

  • Secondary structure

  • Sweet spot: 18-30 nucleotides

(primer design) melting point of primer

• Ideally between 60-65 degrees C

• Melting temp of primers should be within 2 C of each other

• Use an annealing temperature that is about 5 deg lower than the lowest Tm in the primer pair

• Melting temp calculation/equation: Tm in C = 4(#C+#G) + 2(#A+#T)

melting point/temp (Tm)

temperature at which half of DNA duplex dissociate into single strands; temp where the primer and template start to come apart

practice problem (see pic)

• 1st strand: Tm C = 4(7+4) + 2(5+3) = 60

• 2nd strand: Tm C = 4(5+5) + 2(5+5) = 60

• Answer: would make a good pair

(primer design) complementary primer sequences

• complementation within a single primer = hairpin

  • more than 3 base pairs = primer can binds to itself and not to template

• complementation between primers

  • primers anneal to each other forming a primer dimer (DNA pol extends the primer and not actual template)

primer design process

• Identify priming sites flanking a region/gene/variant of interest using NCBI gene database

• Using a primer design software (e.g. Primer 3) and primer parameters, identify potential primers

• Check primer specificity by in silico PCR

• Check for SNPs under the primers

  • Primer may not bind as well

(optimizing PCR) annealing temp

• Too low: products are more nonspecific; primers bind to things they aren't supposed to--bind randomly to sequences that aren't perfectly complementary

• Too high: primers can't bind

• Gradient protocols: programming the thermal cycler for a range of temperatures across the heat block, allowing for the ID of optimal annealing temperature

(optimizing PCR) hot start

• Hot start polymerase: a sequestered enzyme that requires an initial heat activation

  • Benefits: starts the reaction at desired time and temp; avoid getting misprimes and unwanted products

• If NOT using a hot start enzyme, reactions should be set up on ice

(optimizing PCR) additives

• Can increase PCR yield and specificity

  • Dimethyl sulfoxide (DMSO)--use for when you have lots of C-Gs; prevents secondary structures in the DNA templates with more C=Gs

  • Formamide

  • Nonionic detergents

  • Polyethylene glycol

  • Bovine serum albumin

  • N,N,N-trimethyl-glycine (betaine)

multiplex PCR

• Simultaneous amplification of multiple targets in a single reaction

• Each target requires a separate primer pair

• Requires extensive optimization for successful amplification of all targets

• Differentiation of resulting amplicons:

  • Amplicon size

  • Fluorescently-labeled primers

  • Fluorescently-labeled probes

(PCR applications) Factor V Leiden

• Point mutation in the Factor V gene

  • Arginine (CGA) → Glutamine (CAA) @ Arg 506

• Inherited FVa resistance to APC cleavage

• Prevalence in European ancestry ~3-8%

• Autosomal dominant trait

• Responsible for ~60% of familial thrombosis

factor V leiden molecular diagnosis

method: Restriction fragment length polymorphism (RFLP): some mutations change a restriction enzyme site

• PCR to amplify sequence of interest

• Digest protein with enzyme of interest

• Detect normal and mutant sequences with electrophoresis

(PCR applications) hereditary hemochromatosis (HH)

• Autosomal recessive disorder where mutations in HFE gene = increased intestinal iron absorption and deposition of excessive amount of iron into the liver, pancreas, and other organs

• HH is the most common genetic disorder in individuals of European ancestry

  • Heterozygote carriers: about 10%

  • Homozygous: 1 in 250 to 300

clinical features of hereditary hemochromatosis

• hyperpigmentation, diabetes mellitus, liver cirrhosis

• Other clinical features

  • Fatigue

  • Hepatomegaly (elevated serum aminotransferase)

  • Arthropathy

  • Hypogonadism (impotence in males)

  • Hypothyroidism

  • Cardiomyopathy

• Symptoms typically occur late in the disease, when the total body iron content has reached as high as 20g (more than 5x normal)

• Most patients are diagnosed earlier, when elevated serum iron levels are noted on a routine screening panel or screening is performed because of a relative diagnosed with HH

• Approx 75% of pts are asymptomatic at presentation

hereditary hemochromatosis genetics

• Affected gene: HFE

  • HFE protein interacts with cell surface proteins to detect amount of iron

  • HFE protein regulates hepcidin, which regulates iron

  • Two point mutations associated with HH:

    • A cysteine to trypsin substitution at AA 282 (C282Y)

    • A histidine to aspartic acid substitution at AA 63 (H63D)

hereditary hemochromatosis molecular diagnostics

• Two separate PCR-RFLP analyses using primers flanking C282Y or H63D

• Molecular diagnostics cannot provide information about the degree of increased body iron stores or organ damage and thus cannot replace liver biopsy

(PCR applications) STR testing

• STR = short tandem repeat = microsatellite = simple sequence repeat (SSR)

• Area or DNA where a short pattern of nucleotides is repeated multiple times

• Individuals vary in how many repeats are present

• types of STR repeat units (pic)

(STR testing) why can’t regular agarose gel be used for this kind of testing?

agarose gel does not have enough resolution ; need to be able to tell the difference between things that are only 1-2 bp apart

STR testing (general)

• Multiplex PCR used to copy several STR loci at once

• Sensitivities to levels less than 1 ng of DNA

• Ability to handle mixtures and degraded samples

• Primers labeled with fluorescent dyes

• Different fluorescent dyes used to distinguish STR alleles w overlapping size ranges

(STR testing) FBI CODIS DNA database

• Used for linking serial crimes and unsolved cases w repeat offenders

• Missing person/unidentified human remains

• Launched October 1998

• Links all 50 states

• Requires 20 STR markers--13 original "core" + 7 new

other uses for STR testing

• Forensic paternity

• Chimerism

  • Allogenic bone marrow transplants are monitored using STR

  • two parts of chimerism testing: pretransplant informative analysis and post-transplant engraftment analysis

modifications of PCR (list)

• Multiplex PCR

• Sequence specific PCR

• Reverse transcriptase PCR

• Nested PCT

• Real-time (Quantitative) PCR)

sequence specific PCR

• In regular PCR, the primer can still bind to the template even if there are some mismatches

• However, the primer MUST be complementary at the 3' end for annealing to occur

• Can use to ID single base changes in DNA

• Make primer that is complementary to mutant sequence at 3' end

  • Normal sequence = no amplification

  • Mutant sequence = yes amplification

    • Can detect mutant sequences; can go the other way too

reverse transcriptase PCR

• use when starting material is RNA (viruses/diseases with errors in gene expression)

• Use an enzyme called reverse transcriptase to turn RNA into DNA

  • Requires primers to generate DNA from RNA (oligo dT or random hexamers typically)

• This results is a double stranded DNA copy of the RNA called cDNA

nested PCR

• Increases specificity and sensitivity

• Two pairs of primers are used in two separate runs

• First pair of primers--amplify sequence of interest

• Second pair--binds slightly inside of first pair

• How would this increase sensitivity and specificity?

  • Sensitivity = allows for more cycles to be run

  • Specificity = corrects for misprimes and other issues when the cycles are run; binding of two separate sets of primers to the same target template

real time PCR (general)

• not only tells us if a sequence is present, but how much

• Detects the accumulation of product during exponential phase which can be monitored in real time

• No gel-based analysis at end of reaction

• Computer based analysis based on fluorescence detection

standard PCR vs real-time PCR

• Standard (qualitative)

  • Detects if disease is there or not

• RT-PCT (qualitative/quantitative)

  • Viral load

  • Bacterial load

  • Amount of gene expression

(terminology) RT-PCR

• RT-PCR: could refer to "real-time" PCR = DNA amplification is monitored during PCR process

• RT-PCR: could also refer to "reverse transcriptase" PCR = method to detect RNA

(terminology) qPCR

"quantitative" PCR functionally the same thing as real-time PCR

(terminology) qRT-PCR

most likely refers to quantitative reverse transcriptase PCR = quantitating the amount of RNA present

(real-time PCR) chemistry + detection + analysis method

• Chemistry: use fluorescent dyes, probes, or primers

  • Establish a linear correlation between PCR product and fluorescence intensity

• Detection: fluorescence detection to monitor amplification monitored in real time

• Analysis: software analysis and estimate of template concentration

(real-time PCR) Ct value

cycle number at which the fluorescence intensity crosses the threshold line

• if a sample has MORE starting material it will have a LOWER Ct value compared to something with less starting material

• takes less cycles for it to cross the threshold

(real-time PCR) how to set a threshold

• Set within the exponential phase of PCR

• Needs to be set above background "noise"

(real-time PCR) standard curves

• Serial dilution series of sample with known number of copies

• Use this to establish the relationship between starting target copy number and Ct (cycle number)

(real-time PCR) example qPCR standard curve problem: what is the starting number of copies of DNA in each sample?

• Sample 1 Ct = 25

• Sample 2 Ct = 18

• Sample 3 Ct = 10

• Sample 1: 12,148

• Sample 2:  1,157,746

• Sample 3: out of linearity; dilute and rerun, multiply by dilution factor

(real-time PCR) efficiency

• How well a reaction doubles the target DNA sequence in each cycle

• How to measure efficiency:

  • Perform a 10-fold dilution series

  • Generate a standard curve of log copy number vs. CT

  • E = -1+10^(-1/slope)

  • Slope of 100% efficient reaction is -3.33

    • i.e., there are 3.33 cycles between CT values of each 10-fold dilution

(real-time PCR) what should the value for efficiency be?

• in between 90 and 100%

• Slope should be between -3.6 and -3.3

• If below -3.6, then rxn has poor efficiency

• Causes of poor efficiency:

  • Samples may contain PCR inhibitors

  • PCR primer and/or probe design not optimal

  • Inaccurate sample and reagent pipetting

  • Standard curve not properly analyzed

(real-time PCR) qPCR negative controls

• NTC (no template control): should be negative—if positive, you may be detecting primer dimers

• NAC (no amplification control): omit DNA polymerase—controls for background fluorescence

• NRT (no reverse transcriptase): makes sure no genomic DNA is present when your starting material was RNA

(real-time PCR) qPCR positive controls

• Exogenous positive control: external DNA or RNA carrying a target of interest

• Endogenous positive control: target that is present in the experimental sample(s) of interest, but is different from the target under study (typically a "housekeeping" gene)

(real-time PCR) Your no template control is positive. What could be the cause? How would you resolve?

contamination/primer dimers

• use different primers/different temp or run PCR again

(real-time PCR) Your endogenous control (positive) is negative. What could be the cause? How would you resolve?

bad sample/missed step or reagent/bad reagent

• recollect, run again OR add reagent/replace reagent

(real-time PCR) melt curves

• Something that you can add on after amplification is complete

• Typically used with SYBR green chemistries

• Answers if you only amplify one product or not

• Slowly heat the end product--as DNA denatures, the fluorescent signal goes away

• Larger products = more heat to denature

(real-time PCR) technical replicates

• Running multiple reactions on the sample (usually 3)

• qPCR highly sensitive to pipetting errors

• Ideally replicates should be tightly clustered

(real-time PCR) ideal run graph (pic)

• curves are all S shaped

• dilution series has expected spacing

• replicates are tightly clustered

• baselines are relatively flat

• plateau height doesn’t matter

• curves are smooth

• melt curve has one peak per product

(real-time PCR; troubleshooting) replicates

• If replicates aren't tightly clustered, suspect:

  • Pipetting error

  • Poorly optimized PCR reactions

  • Sample evaporation

  • Unknowns outside of range of detection

  • Instrument calibration

(real-time PCR; troubleshooting) baselines

• If baselines aren't flat, suspect:

  • Sample evaporation

  • Bubbles

  • Reagents not thoroughly mixed

  • Baseline "window" not properly set

(real-time PCR; troubleshooting) dilution series

• If the dilution series comes out "compressed" or "stretched," suspect:

  • Pipetting

  • Too much DNA (for your assay)

  • PCR inhibitors 

  • Too little DNA (for your assay)

  • Poor PCR efficiency

(real-time PCR; troubleshooting) curve shape

• If curves are not S-shaped, suspect:

  • Curves are not actual PCR products!

  • Sample evaporation

  • Fluorescence drift in unamplified samples

  • Something seriously wrong with assay

• If curves are not smooth, suspect:

  • Poor pipetting (bubbles)

  • Sample evaporation

  • Poor assay (low fluorescence reagents)

  • System malfunction

(real-time PCR; troubleshooting) melt peaks

• If melt curves have more than one peak:

  • More than one product

  • Possible normal primer-dimer

  • Using too low an annealing temperature

  • Primers need to be redesigned

advantages of qPCR

• Quantitative

• Amplification and detection all in one

• No post-PCR processing

• Reproducible

• Potentially not influenced by non-specific amplification

  • Many chemistries are sequence specific or use melt curve analysis

• Rapid

• Very sensitive

disadvantages of qPCR

• A more limited capacity for multiplexing than standard PCR

• Development of protocols needs high level of technical skill and/or support (requires R&D capacity and capital)

• High capital equipment costs

(qPCR applications) HCV

• Blood borne virus

• Initial infection is often asymptomatic, but can result in chronic infection leading to cirrhosis of the liver and liver cancer

• No vaccine ; curative treatment = direct acting antivirals

• diagnosed through reverse transcriptase and qPCR

HCV reverse transcriptase-PCR viral quantitation detection

• Convert the RNA to DNA using reverse transcriptase

  • An HCV-specific primer is used to prime the cDNA synthesis reaction

• After cDNA synthesis, a PCR reaction is performed using HCV-specific primers

  • Taq DNA polymerase

  • Two primers: the primer used for cDNA synthesis and a second HCV-specific primer

how does real-time PCR detect the formation of products?

fluorescence (SYBR green, TaqMan, FRET, Molecular Beacon, isoG:C, Scorpion)

fluorescence

• Absorption of light cause excitation of electrons

• When electrons return to the ground state, light is emitted--fluorescence

• Light emitted is always lower energy (longer wavelength) than light absorbed

(qPCR chemistries) SYBR green

• SYBR green is a fluorescent dye that binds to dsDNA

• Low fluorescence when not bound to dsDNA

• Higher levels of fluorescence with DNA binding

• Absorption maxima 497 nm (blue)

• Emission maxima 520 nm (green)

what stage of qPCR is fluorescence detected with SYBR green?

during extension! (last stage)

• little to none during denaturation

• little bit during annealing

when is maximal fluroescence detected with SYBR green? when is the measurement taken?

• at the end of extension step

• taken at the end of a cycle/extension

pros/cons to SYBR green

pros

• Easy ; cheap

• No need to design/buy probes

cons

• Nonspecific

• More background fluorescence compared to other methods

 

types of probe-based detection chemistries (3)

• TaqMan

• FRET probes

• Molecular Beacons

probe

a single stranded synthesized DNA molecular, often about 18-25 bases in length

• Complementary to target—anneals between primer binding sites

• Labeled with fluorescing and quenching nucleotides

probes vs primers

• probes are NOT designed to be the starting place for Taq pol—no 3’ end to attach bases to

• usually only one probe is used compared to 2 primers

(probe based detection) fluorescence resonance energy transfer

• aka FRET ; basis behind most probe technologies

• Reporter (R) absorbs light (blue) and fluoresces (green)

• Emitted green light is absorbed by the Quencher (Q)

• Quencher then emits light of still longer wavelength (red)

• This energy transfer only occurs when the Reporter is next to the Quencher

(FRET) quenching nucleotides

• Absorb the energy emitted by fluorescing nucleotides

  • Light quenchers: absorb fluorescent energy from the "reporter fluor" and then emits fluorescent light at a longer wavelength

  • Dark quenchers: absorb fluorescent energy from the "reporter fluor" and then do not emit light

(probe based detection) TaqMan probes

• Unbound probe free in solution (no reporter fluorescence)

• During annealing, probe and primer bind target (no reporter fluorescence)

• During extension, Taq hydrolyzes (eats up) probe and reporter can now emit fluorescence

when is the fluorescent signal detected in TaqMan probe based methods?

during extension!

• Taq pol will hydrolyze the probe and the reporter/quencher are now unbound and far away so the quencher doesn’t absorb up all the signal

(probe based detection) FRET probes

• uses two adjacent hybridization probes:

  • One attached to donor fluorophore (reporter)

  • One attached to accepter fluorophore (quencher)

  • Designed to bind near each other

• No signal from the acceptor fluorophore without binding near the donor fluorophore on the DNA template

• When they bind near each other, the donor fluorophore excites the acceptor fluorophore which then emits fluorescence that can be measured

at what point in the PCR cycle should fluorescence be measured for FRET probes?

at the annealing step

• probes can be resused and are not destroyed

(probe based detection) molecular beacons

• Hairpin shaped DNA probes

• Labeled with reporter and quencher at 5' and 3' end

• Added to PCR reaction mix

• Hybridize to target during PCR annealing step

• Hybridization physically separates fluor from quencher allowing reporter fluorescence

• Maximal fluorescence during ANNEALING

what two chemistries is the signal gathered at the annealing step?

• FRET probes

• Molecular Beacons

(fluorescently labeled chemistries) isoG:C

• Forward primer labeled at 5' end with fluorescent iso-C

• Reverse primer is unlabeled

• Dabcyl-iso-dGTP (quencher) in dNTP mix

• During elongation, dabcyl-iso-dGTP incorporated opposite iso-C

• Proximity of dabycl to reporter quenches fluorescence

• Signal decreases as product accumulates

(fluorescently labeled chemistries) scorpion primer/probes

• Similar to molecular beacons but a primer and a probe are contained in one molecule

• After extension of the primer, the target-specific sequences fold over to hybridize with the newly synthesized target sequences, separating the reporter from the quencher

list of real-time PCR applications

• Pathogen detection and genotyping assays

• Microbial/viral load assays

• SNP detection, allele discrimination, genotyping, haplotyping

• Clinical diagnostics (cancer, therapy response)

• DNA target quantification (nuclear, mitochondrial, residual DNA in protein preps [QC])

• Gene expression (and microarray validation)

• DNA methylation, apoptosis analysis

(qPCR applications) HSV detection

• can make FRET probes to HSV-1 and HSV-2

• if the probes attach to the amplicon DNA without a mismatch, then HSV-1 is suspected

• HSV-2 is suspected when there is a mismach in the FRET probes

• can be confirmed with a melt curve (measuring the change in fluorescence)

  • due to the probe having a mismatch with HSV-2, the probe will dissociate from the product at lower temperatures

digital droplet PCR

• Digital PCR that is based on water-oil emulsion droplet technology

• Each droplet is read in a flow cytometer to determine the positive droplets in sample

• Data is then analyzed using Poisson statistics to determine the target DNA template concentration

• Provides and absolute quantification of target DNA copies without need for a standard curve

• Particularly useful for detecting a small number of tumor cells among lots of wild-type cells/rare mutations